Mutations in chromatin regulatory proteins have recently been identified in a wide range of human cancers. Although these mutations are associated with disease progression, how they contribute to tumorigenesis remains unknown. We propose to investigate SETD2, a non-redundant histone methyltransferase that preferentially places a trimethylation mark on Histone H3 on lysine 36 in actively transcribed gene bodies. This gene is mutated in approximately 15% of renal cell carcinomas, and is being identified in a growing list of tumors. By examining chromatin organization in primary human tumors, we observed that SETD2 mutation and loss of its histone modification were associated with changes in nucleosome accessibility and widespread alteration in RNA processing. We hypothesize that histone mark dysregulation through SETD2 loss is an important component of tumorigenesis. We propose a highly collaborative project that employs biochemical, genetic and genomic approaches to comprehensively explore the effect of SETD2 mutation on chromatin and transcription, in an effort to unravel the mechanisms by which SETD2 loss leads to the promotion of cancer. To this end, we have generated a unique set of cell-based model systems which uses comparative genomic studies between human and yeast cells. We have additionally established novel domain-specific activities of SETD2, and the first demonstration of functional mutations separating di-methylating from tri-methylating activity. We propose three complementary aims: 1) to define the chromatin reprogramming and transcriptional effects of SETD2 mutation, revealing critical mechanistic features that link histone modification and nucleosome position to the transcriptional and DNA repair defects associated with SETD2 loss, 2) to utilize the separation of function revealed by high severity SETD2 mutants in the catalytic (SET) domain and the RNA polymerase II interactions domain (SRI) to reveal important determinants of the genomic phenotype, and 3) to explore discrete functions associated with SETD2 loss mediated by the altered H3K36me3 mark, by systematically examining the histone reader molecules that transduce signals for DNA methylation, DNA repair, and transcriptional elongation. Taken together, these studies will define the mechanistic link between SETD2 loss of function and cancer development, as well as reveal novel opportunities for biomarker or therapeutic interventions.